Rhabdomyosarcoma (RMS) is a myogenic cancer that is the most common soft tissue sarcoma of childhood. With the development of multimodal chemotherapy regimens, relapse-free survival rates have improved to 70-80% in patients with localized disease, albeit with significant toxicity. Unfortunately, despite aggressive therapy, the 5 year survival rate for patients with metastatic disease remains only 30%. Therapy assignment is currently based on clinicopathologic features and using these criteria, three distinct subgroups of patients can be identified (low, intermediate and high risk). However, many patients fall into the intermediate risk category (which accounts for about 50% of all patients) and have a heterogeneous clinical outcome. This suggests that some of these children could be treated with less aggressive therapy or alternatively should be considered to have more aggressive disease. In an effort to further characterize the genetic events underlying this tumor type, our group in collaboration with the Children's Oncology Group performed a large sequencing effort using a combination of whole-genome, whole-exome and whole-transcriptome sequencing along with high resolution SNP arrays to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Our findings describe a heterogenous group of genetic events appears to drive RMS most notably the PAX 3/7-FOXO1 fusion in the alveolar subtype and mutation of multiple RAS pathway genes in fusion negative tumors including recurrent genetic lesions in 10 cancer consensus genes (NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1). While the majority of these mutations appear to be mutually exclusive, a subset of tumors appears to have coexisting lesions within the same tumor; perhaps indicating a biologically relevant progression in these tumors. Unfortunately, much of the clinical annotation for these cases was incomplete, severely limiting our ability to derive prognostic information from this data set. To overcome this, a more focused retrospective analysis is needed to determine the prognostic significance of the discovered mutations. In collaboration with the Children's Oncology Group we have identified 675 clinically annotated rhabdomyosarcoma cases. In this project we are using high throughput sequencing to determine the frequency with which the observed mutations occur and determine whether these mutations could be used as markers of prognosis or response to therapy. The specific objectives of the study are the following: 1. To validate our pilot study and accurately estimate the frequency of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in low, intermediate and high risk embryonal rhabdomyosarcomas. 2. To estimate the frequency of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in a small cohort of alveolar histology tumors. 3. To determine the clinical significance of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in patients with central pathology confirmed ERMS cases. 4. To develop an accurate and reproducible assay for assessing mutations in NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 that can be used in prospective clinical trials. Within the past year, we have built a pipeline for the extraction of nucleotides, targeted gene capture and sequencing and bioinformatic analysis of the data. We have validated that our assay can reliably detect mutations even at low frequency and that we can accurately detect gene copy number changes. We have completed sequencing and data analysis of 300 cases to date. We have developed a bioinformatics pipeline and visualization portal that enables a user friendly interface with the generated data. We have initiated clinical outcome correlations with the presence or absence of each genetic lesion. In the second objective of the project, we are using high-throughput drug screening to discover novel therapeutics that specifically inhibit the fusion oncogene PAX3-FOXO1. PAX3-FOXO1 is a genetic change that is associated with particularly bad clinical outcomes. Within the past year, we have built and optimized a cell line construct that can robustly measure the transcriptional output of the PAX3-FOXO1 fusion gene. We have completed the primary screen of approximately 125,000 compounds and natural product extracts. Our cell line construct was found to perform reliably in the assay and the generated data has been verified. From the primary screen we identified approximately 200 candidate compounds or mixtures that specifically inhibit the transcriptional output of PAX3-FOXO1 in a dose response manner. We have developed a secondary screen that can rapidly asses additional downstream targets of the fusion gene and have used RNA sequencing to globally characterize the transcriptional effects of our lead candidate compounds. We are currently functionally characterized three lead compounds from the screen using in vitro and in vivo models of rhabdomyosarcoma.